In the production of recombinant proteins in heterologous organisms such as the expression of human or other eukaryotic proteins in bacterial cells it is often difficult to obtain a clearly defined N-terminus which is as nearly 100% homogeneous as possible. This apples in particular to recombinant pharmaceutical proteins whose amino add sequence in many cases ought to be identical to the amino acid sequence naturally occurring in humans/animals.
On natural expression, for example in humans, many pharmaceutical proteins which are in use for therapy as well are transported into the extracellular space. A signal sequence is present in the precursor protein for this purpose and cleavage of this signal sequence results in a clearly defined N-terminus. For several reasons such homogeneous N-termini are not always easy to produce, for example in bacterial cells.
For production on industrial scale, many pharmaceutical proteins are produced in the cytoplasm of bacterial cells (for example Escherichia coli). In the host cells the pharmaceutical proteins are accumulated in adequate quantities and are often deposited as Insoluble inclusion bodies (IBs) inside the cell. These IBs have great advantages in working up and purification of the target protein. In addition, the protein expressed in the form of IBs is protected from protease degradation by intacellular proteases.
As used herein the term “inclusion bodies” shall refer to aggregates containing heterologous polypeptides present in the cytoplasm of transformed host cells. These appear as bright spots under the microscope and can be recovered by separation of the cytoplasm.
However, production of IB material requires in vitro refolding of the expressed protein. This can in many cases be effected by methods known per se.
Only in rare cases is export of the target protein into the bacterial periplasm with the aid of a pro or eukaryotic signal sequence suitable. Because of the low transport capacity of the bacterial export machinery it is usually only possible to accumulate very small quantities of product here.
However, the bacterial cytoplasm differs considerably from the extracellular space of eukaryotes. One difference is that within the bacterial cytoplasm reducing conditions are predominant, also a mechanism for cleaving N-terminal leader sequences to form mature proteins is lacking. Synthesis of all cytoplasmic proteins starts with a methionine which is specified by the appropriate start codon (ATG=initiation of translation). This N-terminal methionine is retained in many proteins, while in others it is cleaved by the methionine aminopeptidase (MAP) present in the cytoplasm and intrinsic to the host. The efficiency of the cleavage depends essentially on two parameters: 1. the nature of the following amino acid, and 2. the location of the N-terminus in the three-dimensional structure of the protein. The N-terminal methionine is preferentially deleted when the following amino acid is serine, alanine, glycine, methionine or valine and when the N-terminus is exposed, i.e. not “hidden” inside the protein. If the following amino acid is a different one, in particular a charged one (glutamic acid, aspartic acid, lysine, arginine), or if the N-terminus is located inside the protein, in most cases cleavage of the N-terminal methionine does not occur.
Even if an amino acid that promotes cleavage is present at position 2, the cleavage is rarely complete. It is usual for a not inconsiderable portion (1-60%) of the target protein to remain unaffected by the MAP.
This in-homogenelty or deviation from the natural sequence is, however, unacceptable in many cases because these products frequently show different immunological (for example induction of antibody formation) and pharmacological (half-life, pharmacokinetics) properties. For these reasons, it is necessary in most cases to produce a nature-identical product (homogeneous and without foreign amino acids at the N-terminus). In the case of cytoplasmic expression, the remedy here in most cases is to fuse a cleavage sequence (leader) for a specific endopeptidase (for example factor Xa, enterokinase, KEX endopeptidases, IgA protease) or aminopeptidase (for example dipeptidyl aminopeptidase) to the N-terminus of the target protein. However, this makes an additional step necessary during further working up, the so called down stream processing of the protein, with expenditure of costs and materials. In addition, in the presence of IBs there is in many cases interference with or even complete prevention of the refolding by the leader sequence.
Fusion polypeptides comprising the autoprotease Npro of Pestivirus are especially useful in this respect. The autoprotease Npro of Pestivirus always cleaves off the fusion partner at a clearly determined site, releasing a polypeptide of interest with homogenous N-terminus. In addition, the autoproteolytic activity of Npro can be induced in vitro, by application of special buffers, so that the polypeptide of interest can be obtained by cleavage of fusion polypeptides that are expressed in IBs.
Pestiviruses are small enveloped viruses with a genome which acts directly as mRNA and is 12.3 kb in size and from which the viral gene products are transcribed in the cytoplasm. This takes place in the form of a single polyprotein which comprises about 4000 amino acids and which is broken down both by viral and by cellular proteases into about 12 mature proteins.
Pestiviruses comprise the subclasses CSFV (classical swine fever virus), BDV (border disease virus) and BVDV (bovine viral diarrhoea virus).
Npro is an autoprotease with a length of 168 amino acids and an apparent Mr of about 20,000 (in vivo). It is the first protein in the polyprotein of Pestiviruses and undergoes autoproteolytic cleavage from the following nucleocapsid protein C. This cleavage takes place after the lest amino add in the sequence of Npro, Cys168.
Use of the naturally occurring autoprotease Npro of Pestivirus for production of heterologous polypeptides of interest may be limited though, as activation of autoproteolytic function of Npro in vitro is susceptible only to specific renaturazing conditions. These conditions that allow for the cleavage activity of Npro in vitro are inhibitory for certain other interactions which are necessary or desirable in some settings for production of heterologous polypeptides of interest. As an example of such interactions certain bio-specific affinities as e.g. selective peptide-protein affinity can be named. Also, due to other requirements of parameters, certain processes do not permit to create the favourable renaturazing conditions for Npro and as a result Npro can not be used in these processes. Therefore the naturally occurring Npro of Pestivirus may be unsuitable for the production of certain polypeptides of interest and for use under certain conditions. Accordingly the need for an Npro of Pestivirus with improved properties exists, in order to enhance cleavage efficiency, to obtain higher yields of polypeptide of interest, and in order to be able to use Npro in a wider range of conditions, which allow for the application of new production processes.